Abstract

CpG DNA, a ligand for Toll-like receptor 9 (TLR9), has been one of the most promising immunotherapeutic agents. Although there are several types of potent humanized CpG oligodeoxynucleotide (ODN), developing "all-in-one" CpG ODNs activating both B cells and plasmacytoid dendritic cells forming a stable nanoparticle without aggregation has not been successful. In this study, we generated a novel nanoparticulate K CpG ODN (K3) wrapped by the nonagonistic Dectin-1 ligand schizophyllan (SPG), K3-SPG. In sharp contrast to K3 alone, K3-SPG stimulates human peripheral blood mononuclear cells to produce a large amount of both type I and type II IFN, targeting the same endosome where IFN-inducing D CpG ODN resides without losing its K-type activity. K3-SPG thus became a potent adjuvant for induction of both humoral and cellular immune responses, particularly CTL induction, to coadministered protein antigens without conjugation. Such potent adjuvant activity of K3-SPG is attributed to its nature of being a nanoparticle rather than targeting Dectin-1 by SPG, accumulating and activating antigen-bearing macrophages and dendritic cells in the draining lymph node. K3-SPG acting as an influenza vaccine adjuvant was demonstrated in vivo in both murine and nonhuman primate models. Taken together, K3-SPG may be useful for immunotherapeutic applications that require type I and type II IFN as well as CTL induction.

K3-SPG acts as a potent vaccine adjuvant by simple mixture with antigen. Adjuvant activities of K3-SPG were analyzed. C57BL/6J mice (n = 4 or 5) were immunized s.c. with OVA protein antigen and various adjuvants. OVA-specific serum IgG (A), IFN-γ (B), and OVA257–264-specific tetramer (C) were monitored (d17) after immunization (d0 and d10) with OVA (100 μg) with or without K3 (10 μg) or K3-SPG (10 μg). (D) In vivo CTL assay 7 d after priming with OVA and various adjuvants as indicated. (E) Immunization with OVA257–264 peptide (10 μg) with or without adjuvant as indicated. (F and G) Dose-sparing study; OVA-specific serum IgG and IFN-γ were monitored after immunization as in A and B. *P < 0.05 (Mann–Whitney U test). Data represent one of two or three independent experiments with similar results.

Adjuvant effects of K3-SPG were completely dependent on TLR9 and partially on Dectin-1. FL-DCs (A and C) or splenocytes (B and D) from C57BL/6J, Tlr9−/−, or Dectin-1−/− mice were stimulated with K3-SPG [20 μg/mL (A), 0.014–10 μg/mL (B), or 0.014–10 μg/mL (C and D)] for 24 h, and their cytokine production was monitored by ELISA. Tlr9+/− (n = 7) or Tlr9−/− mice (n = 10) (E–G) and Dectin-1+/− (n = 5) or Dectin-1−/− mice (n = 6) (H–J) were immunized s.c. with OVA (100 μg) and K3-SPG [10 μg (E–G) or 1 μg (H–J)] at days 0 and 10. Seven days after the last immunization, OVA-specific serum IgG (E and H), IFN-γ (F and I), and OVA257–264-specific tetramer (G and J) were monitored. *P < 0.05 (Mann–Whitney U test). Data represent one of two or three independent experiments with similar results.

Role of lymph node macrophages and dendritic cells in uptake and adjuvant effects of K3-SPG. (A) Immunohistochemistry of mouse inguinal LNs after Alexa 488-K3-SPG injection. One hour after injection, the LNs were collected and stained with anti–MARCO-phycoerythrin (PE) and anti–Siglec-1-APC antibodies. (B–E) Two-photon microscopic analysis of LNs. DQ-OVA, Alexa 488-K3, or Alexa 488-K3-SPG was injected as indicated, and anti–MARCO-PE or anti–Siglec-1-PE antibodies were administered. The LNs were collected 1 h later and analyzed by two-photon microscopy. (C and E) Colocalization of antigen or adjuvant with the stained macrophages was analyzed by Pearson’s correlation. (F) Clodronate liposomes were injected into C57BL/6J mice either 2 or 7 d before immunization (n = 4). Mice were administered OVA (100 μg) plus K3-SPG (10 μg) at day 0. Eight days after immunization, OVA-specific serum IgG and IFN-γ were monitored. (G) C57BL/6J and Tlr9−/− mice were administered s.c. with K3 (10 μg) or K3-SPG (10 μg). At 24 h postadministration, the LNs were collected and the prepared cells were stained and analyzed by flow cytometry. (Scale bars, 100 μm.) *P < 0.05 (t test or Mann–Whitney U test). Data represent one of two or three independent experiments with similar results.